The commitment of barley microspores into embryogenesis involves miRNA-directed regulation of members of the SPL, GRF and HD-ZIPIII transcription factor families

SUMMARYMicrospore embryogenesis is a model for developmental plasticity and cell fate decisions. To investigate the role of miRNAs in this development, we sequenced sRNAs and the degradome of barley microspores collected prior to (day 0) and after (days 2 and 5) the application of a stress treatment known to induce embryogenesis. Microspores isolated at these timepoints were uniform in both appearance and in their complements of sRNAs. We detected 68 miRNAs in microspores. The abundance of 51 of these miRNAs differed significantly during microspore development. One group of miRNAs was induced when the stress treatment was applied, prior to being repressed when microspores transitioned to embryogenesis. Another group of miRNAs were up-regulated in day-2 microspores and their abundance remained stable or increased in day-5 microspores, a timepoint at which the first clear indications of the transition towards embryogenesis were visible. Collectively, these miRNAs might play a role in the modulation of the stress response, the repression of gametic development, and/or the gain of embryogenic potential. A degradome analysis allowed us to validate the role of miRNAs in regulating 41 specific transcripts. We showed that the transition of microspores toward the embryogenesis pathway involves miRNA-directed regulation of members of the ARF, SPL, GRF and HD-ZIPIII transcription factor families. We noted that 41.5% of these targets were shared between day-2 and day-5 microspores while 26.8% were unique to day-5 microspores. The former set may act to disrupt transcripts involved in pollen development while the latter set may drive the commitment to embryogenesis.

The mitochondrial aldehyde dehydrogenase OsALDH2b negatively regulates tapetum degeneration in rice

Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Although several genes involved in tapetum development have been identified, the molecular basis of tapetum degeneration regulation remains poorly understood. In this study, we identified and characterized the nucleus-encoded, conserved mitochondrial aldehyde dehydrogenase OsALDH2b as a key regulator of tapetum degeneration in rice (Oryza sativa). OsALDH2b was highly expressed in anthers from meiosis to the early microspore stage. Mutation of OsALDH2b resulted in excess malonaldehyde accumulation and earlier programmed cell death in the tapetum, leading to premature tapetum degeneration and abnormal microspore development. These results demonstrate that OsALDH2b negatively regulates tapetal programmed cell death and is required for male reproductive development, providing insights into the regulation of tapetum development in plants.

Pollen Viability and Microspore Culture in Three Broccoli Cultivars (Brassica oleracea L. var. italica Plenck)

Broccoli is a high value vegetable crop in Indonesia, however production is low due to limited number of suitable cultivars, so, breeding hybrid broccoli for warm climate is important. The first step in hybridization is providing homozygote parent plants which can be done efficiently via microspore culture. The objectives of this study were to determine 1) bud size that produce uninucleate microspore stage appropriate for culture; 2) pollen viability, 3) microspore development, in three broccoli cultivars (‘BL 10001’, ‘Royal Green’ and ‘Green Magic’). Various bud size (1 – 5 cm) was squashed and observed microscopically to determine bud size containing uninucleate microspore. Pollen viability was determined by IKI staining and pollen germination method. Chromosome number was counted on root tips using squash method with aceto-orcein stain. Various heat treatment schemes were conducted to induce microspreo development. Result showed uninucleate microspore derived from 2-3 mm and 3-4 mm bud length of ‘BL-10001’ and ‘Royal Green’ was responsive for microspore development in culture. Pollen viability varied among cultivars, 78-87% on IKI method and 15-16% on germination test. Microspore culture showed different embryogenesis response; pollen-like structure was produced by ‘BL 10001’.

Response of Lilium longiflorum Thunb. Anther with Various Microspore Development Stages in Combinations of Plant Growth Regulator Concentration

The right stage of microspore development to be used as explants become the critical factor for the successful of anther culture. Anther containing microspores at the pollen mother cell to binucleate stages were cultured on Murashige and Skoog (MS) media supplemented with various plant growth regulators. The media of 7.5 mM NAA + 0.75 mM BAP with bud size of 0.6-2.0 cm (pollen mother cell stage) are a combination that fits in callus, indirect shoot and direct shoot initiation with the percentage growth of each 30%; 16.6%; and 14.8%. Chromosome counts of root-tip cell of 89 regenerant revealed that 85 regenerant were diploids (95.5%) and 4 regenerant aneuploids (4.5%), but the haploid regenerants didn’t obtain. This result suggests that regenerants were derived from a somatic cells division.